Woo June Jung

TNF α mediated IL-6 secretion is regulated by JAK/STAT pathway but not by MEK phosphorylation and AKT phosphorylation in U266 multiple myeloma cells.

IL-6 and TNFα were significantly increased in the bone marrow aspirate samples of patients with active multiple myeloma (MM) compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFα than those with MM in plateau phase. TNFα increased interleukin-6 (IL-6) production from MM cells. However, the detailed mechanisms involved in signaling pathways by which TNFα promotes IL-6 secretion from MM cells are largely unknown. In our study, we found that TNFα treatments induce MEK and AKT phosphorylation. TNFα-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKβ or by small interfering RNA (siRNA) targeting TNF receptors (TNFR) but not by MEK, p38, and PI3K inhibitors. Also, TNFα increased phosphorylation of STAT3 (ser727) including c-Myc and cyclin D1. Three different types of JAK inhibitors decreased the activation of the previously mentioned pathways. In conclusion, blockage of JAK/STAT-mediated NF-κB activation was highly effective in controlling the growth of MM cells and, consequently, an inhibitor of TNFα-mediated IL-6 secretion would be a potential new therapeutic agent for patients with multiple myeloma.

Establishment and characterization of bortezomib-resistant U266 cell line: Constitutive activation of NF-κB-mediated cell signals and/or alterations of ubiquitylation-related genes reduce bortezomib-induced apoptosis

Bortezomib has been known as the most promising anti-cancer drug for multiple myeloma (MM). However, recent studies reported that not all MM patients respond to bortezomib. To overcome such a stumbling-block, studies are needed to clarify the mechanisms of bortezomib resistance. In this study, we established a bortezomib-resistant cell line (U266/velR), and explored its biological characteristics. The U266/velR showed reduced sensitivity to bortezomib, and also showed crossresistance to the chemically unrelated drug thalidomide. U266/velR cells had a higher proportion of CD138 negative subpopulation, known as stem-like feature, compared to parental U266 cells. U266/velR showed relatively less inhibitory effect of prosurvival NF-κB signaling by bortezomib. Further analysis of RNA microarray identified genes related to ubiquitination that were differentially regulated in U266/velR. Moreover, the expression level of CD52 in U266 cells was associated with bortezomib response. Our findings provide the basis for developing therapeutic strategies in bortezomib-resistant relapsed and refractory MM patients. [BMB Reports 2014; 47(5): 274-279]

A scientific treatment approach for acute mast cell leukemia: using a strategy based on next-generation sequencing data

Background Mast cell leukemia (MCL) is the most aggressive form of systemic mastocytosis disorders. Owing to its rarity, neither pathogenesis nor standard treatment is established for this orphan disease. Hence, we tried to treat a patient with MCL based on the exome and transcriptome sequencing results of the patients own DNA and RNA. Methods First, tumor DNA and RNA were extracted from bone marrow at the time of diagnosis. Germline DNA was extracted from the patients saliva 45 days after induction chemotherapy and used as a control. Then, we performed whole-exome sequencing (WES) using the DNA and whole transcriptome sequencing (WTS) using the RNA. Single nucleotide variants (SNVs) were called using MuTect and GATK. Samtools, FusionMap, and Gene Set Enrichment Analysis were utilized to analyze WTS results. Results WES and WTS results revealed mutation in KIT S476I. Fusion analysis was performed using WTS data, which suggested a possible RARα-B2M fusion. When RNA expression analysis was performed using WTS data, upregulation of PIK3/AKT pathway, downstream of KIT and mTOR, was observed. Based on our WES and WTS results, we first administered all-trans retinoic acid, then dasatinib, and finally, an mTOR inhibitor. Conclusion We present a case of orphan disease where we used a targeted approach using WES and WTS data of the patient. Even though our treatment was not successful, use of our approach warrants further validation.

Crizotinib in Combination with Everolimus Synergistically Inhibits Proliferation of ALK-Positive Anaplastic Large Cell Lymphoma.

Purpose Anaplastic large cell lymphoma (ALCL) is a rare aggresive non-Hodgkin lymphoma, of which over 50% of cases have an aberrant nucleophosmin (NPM)‒anaplastic lymphoma kinase (ALK) fusion protein. Both mechanistic target of rapamycin (mTOR) inhibitor everolimus and ALK inhibitor crizotinib have shown promising antitumor activity in ALK-positive cancer cell lines. However, their combined effect has not yet been investigated. Materials and Methods We evaluated the anti-proliferative effects of everolimus and/or crizotinib in ALK-positive ALCL cell lines, Karpas 299 and SU-DHL-1, and lung adenocarcinoma cell line, NCI-H2228. Results We found that individually, both everolimus and crizotinib potently inhibited cell growth in a dose-dependent manner in both Karpas 299 and SU-DHL-1 cells. A combination of these agents synergistically inhibited proliferation in the two cell lines. Crizotinib down-regulated aberrant AKT and ERK phosphorylation induced by everolimus. Combination treatment also significantly increased G0/G1 cell-cycle arrest, DNA damage, and apoptosis compared with everolimus or crizotinib alone in ALK-positive ALCL cells. In the Karpas 299 xenograft model, the combination treatment exerted a stronger antitumor effect than monotherapies, without significant change in body weight. The synergistic effect of everolimus and crizotinib was also reproduced in the ALK-positive lung adenocarcinoma cell line NCI-H2228. The combination treatment abrogated phosphoinositide 3-kinase/AKT and mTOR signaling pathways with little effect on the Ras/ERK pathway in NCI-H2228 cells. Conclusion Crizotinib combinedwith everolimus synergistically inhibits proliferation of ALK-positive ALCL cells. Our results suggest that this novel combination is worthy of further clinical development in patients with ALK-positive ALCL.

Abstract 1081: Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model

Background: Despite recent progress in multiple myeloma (MM) treatment with the advent of new targeted anticancer agents such as Bortezomib, Lenalidomide and, Thalidomide MM still remains incurable. To improve clinical outcome of MM patients, many research have been trying to develop novel targeted drugs. Recent studies demonstrated that microRNA Replacement reagent could be a promising therapeutic strategy in patients with hematologic malignancies. Hence, we tried to examine the efficacy of MRX34, a microRNA-34 replacement reagent, in MM animal model. Methods: We examined the anti-tumor activity of a MRX34 against 3MM cell lines (LP1, MOLP-8, and U266), and investigated the survival effects through which this novel agent shows anti-MM activity. For an animal study, we used NRG (NOD-Rag1null IL2rgnull, CB-17 SCID) mice that were injected with MM cells via tail vein. MOLP-8, LP1 and U266 cells were injected Intravenous 106 cells of NRG. When tumors were measurable, mice were assigned to 3 treatment groups receiving the vehicle alone (control), MRX34 0.3mg/kg treatment group and, MRX34 1mg/kg treatment group were given Intravenous 5 times a week. Results: Median survival of mice injected with LP1, MOLP-8 and U266 cell lines were 28days, 29 days and 34days respectively without any treatment. When mice were treated with MRX-34, median survival was 33days, 37days and 42days respectively. There was a significant survival advantage with MRX-34 treatment in MOLP-8 and U266 cell lines. For monoclonal proteins, mice treated with MRX34 group are suppressed M-protein level almost 30% less than M-protein. Summary / Conclusion: Our data indicated that MRX34 had beneficial effects in multiple myeloma animal model. Mice treated with MRX34 showed M-protein suppression and survival advantage. Our results warrant further clinical studies using this novel agent in MM. Citation Format: Woo June Jung, Youngil Koh, Sinil Kim, Hyejoo Park, Sung-Soo Yoon. Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1081.

Abstract 4309: Description of a scientific treatment approach of mast cell leukemia, an aggressive orphan hematologic disorder: strategy based on next-generation sequencing data

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background Mast cell leukemia (MCL) is the most aggressive form of disorder among systemic mastocytosis. Due to its rarity, neither pathogenesis nor standard treatment is not established for this orphan disease. Hence, we tried to treat a patient with MCL based on the exome and transcriptome sequencing result of the patients own DNA and RNA. Brief Case History and Results In October 2013, an 18-year-old Korean female were diagnosed as MCL after visiting our hospital due to left knee, ankle pain and inguinal lymphadenopathy. C-KIT overexpression was observed by immunohistochemistry. Whole exome sequencing result failed to demonstrate either noticeable single nucleotide variant (SNV) or copy number change. Interestingly, whole transcriptome sequencing (WTS) revealed mutation of KIT S476I, functionality of which is not known. Fusion analysis was performed using WTS data, possibility of RARα-B2M fusion has been arised. However, it was not validated by PCR sequencing. When RNA expression analysis was performed using WTS data, upregulation of PIK3/AKT pathway, which is a downstream of KIT (BAD phosphorylation) and mTOR has been observed. For the treatment perspective, she failed to achieve complete remission after cytarabine and idarubicin chemotherapy. Based on our WES and WTS result, we first tried all-trans retinoic acid targeting RARα, which failed to demonstrate efficacy. Then, she received dasatinib targeting KIT, which showed transient response for 2 weeks. Now she is under everolimus targeting mTOR pathway and, further treatment with PI3K inhibitor is planned in case of disease progression. Conclusions We are demonstrating a case of orphan disease, where we used targeted approach using WES and WTS data of the patient. Final results of our treatment outcome will be uncovered shortly, and utility of this kind of approach is to be validated. Citation Format: Jeonghwan Youk, Youngil Koh, Ji-Won Kim, Dae-Yoon Kim, Woo June Jung, Kwang-Sung Ahn, Sung-Soo Yoon, Hye Lim Jung. Description of a scientific treatment approach of mast cell leukemia, an aggressive orphan hematologic disorder: strategy based on next-generation sequencing data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2015-4309

Abstract 1152: Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Substantial advances have been made in understanding the biology of multiple myeloma (MM) through the study of the bone marrow (BM) microenvironment. Dynamic interplay between bone marrow stromal cells (BMSCs) and MM cells regulated the relapsed MM after autologous bone marrow transplantation (ABMT). In this study, we performed gene expression profiling with microarray data to better dissect the molecular phenotypes and prognoses of relapsed multiple myeloma (MM). Using gene expression and clinical data, we applied gene expression signatures reflecting deregulation of oncogenic pathways to highlight molecular changes in bone marrow aspirate from 28 patients with relapsed MM. The patient subgroups were defined according to relapse-free interval, within 6 months versus more than 6 months. The microarray results revealed that dyregulation of MAPK pathway was associated with relapse within 6 months after ABMT. Among them, the expression of PIM-1 gene and MOS gene was higher in samples from patients with relapsed MM than MM cell lines (p=0.0037; p=0.0021). Also, IL-6, sIL-6R, and HGF expression in patients who relapsed within 6 months after ABMT was higher than the patients whose relapse-free interval were longer than 6 months. Treatment of shRNA PIM-1 gene and shRNA MOS gene in U266 and MOLP8 dramatically led to decreasing IL-6/sIL-6R mediated ERK phosphorylation and HGF-mediated MET phosphorylation. Similar results were noted for cluster genes for PIM-1/MOS. Especially, functional analysis of PIM-1 leads to inactivate the MAPK pathway through regulating p38 mediated signaling and Wnt/β-catenin pathway. Recent studies suggested that p38 activity in myeloma inhibits osteoblast differentiation and bone formation, but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Conclusionally, Our analysis suggested that MOS gene and PIM-1, which regulates MAPK pathway, is a noble prognostic marker for relapse of MM. The importance of the PIM-1-MOS-MAPK pathway as a prognostic marker in relapsed MM should be reassessed in the novel therapeutic agent era. Citation Format: Woo June Jung, Kwang-Sung Ahn, Chansu Lee, Youngil Koh, Hyun Jung Lee, Hyo Jung Kim, Hwi-Joong Yoon, Sung-Soo Yoon. Gene profiling of multiple myeloma: MAPK pathway deregulation, which is regulated by PIM-1 and MOS, is associated with relapse within 6 months after autoSCT in MM patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2014-1152

Abstract 1527: CD44v9 involving in multiple myeloma cells adhesion to bone marrow stromal cell promotes bone erosion by augmenting the activation of HGF-receptor/cMet

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The main aim of our study is to determine the significance of the stromal microenvironment in the malignant behavior of multiple myeloma cells. The stroma-derived growth factors/cytokines and hyaluronan act in autocrine/paracrine ways with their receptors, including receptor-tyrosine kinases and CD44 variants (CD44v), to potentiate and support multiple myeloma cell survival. In this study, we found that CD44s and CD44 variants were differentially expressed between fraction of CD138+ fraction and CD138- fraction. Expression levels of CD44v6, CD44v9, and CD44v10, respectively, correlated with bone erosion (p=0.029, p=0.013, p=0.032), suggesting that CD44 variant molecules are involved in multiple myeloma progression. Binding studies using CD44 isoform specific reagents showed that CD44v6 and CD44v9 were involved in binding to bone marrow stromal cells, but not to in vitro synthesized ECM. In 3D culture, CD44v6 and CD44v9-mediated plasma cell binding resulted in a significant induction of HGF secretion by bone marrow stromal cells. CD44v6 and CD44v9-mediated plasma cell binding significantly induces PI3K/Akt via activation the Src-kinase Lyn. In bone marrow serum of MM patients, the expression levels of IL-6, OPN, and hepatocyte growth factor (HGF), respectively, statistically correlated with bone erosion of MM patients (p=0.021, p=0.001, p=0.036). HGF derived from bone marrow stromal cells with multiple myeloma cells stimulates CD44 signaling via activation of HGF-receptor/cMet. Specific CD44 shRNA suppresses HGF-mediated CD44 signaling. Taken together, the role of CD44 variants in adhesion induced HGF- secretion may explain the previously observed correlation between CD44 variants expression and adverse prognosis in multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1527. doi:1538-7445.AM2012-1527